1. Field of the Invention
This invention is in the field of medicinal chemistry. The invention relates to novel piperidinyl compounds and the discovery that these compounds act as blockers of calcium (Ca2+) channels. In particular, the invention relates to a high-throughput screening assay useful for identifying such compounds.
2. Background Art
Calcium ions play fundamental roles in the regulation of many cellular processes. It is therefore essential that their intracellular levels be maintained under strict, yet dynamic control (Davila, H. M., Annals of the New York Academy of Sciences, pp. 102-117 (1999)). Voltage-gated calcium channels (VGCC) serve as one of the important mechanisms for fast calcium influx into the cell. Calcium channels are hetero-oligomeric proteins consisting of a pore-forming subunit (α1), which is able to form functional channels on its own in heterologous expression systems, and a set of auxiliary or regulatory subunits. Calcium channels have been classified based on their pharmacological and/or electrophysiological properties. The classification of voltage-gated calcium channels divides them into three groups: (i) high voltage-activated (HVA) channels, which include L-, N-, P-, and Q-types; (ii) intermediate (IVA) R-type channels; and (iii) low voltage-activated (LVA) T-type channels (Davila, supra). Voltage-gated calcium channels (VGCC) are also known as voltage-dependent calcium channels (VDCC) or voltage-sensitive calcium channels (VSCC).
Voltage-sensitive calcium channels (VSCC) regulate intracellular calcium concentration, which affects various important neuronal functions such as cellular excitability, neurotransmitter release, hormone secretion, intracellular metabolism, neurosecretory activity and gene expression (Hu et al, Bioorganic & Medicinal Chemistry 8:1203-1212 (2000)). N-type channels are found mainly in central and peripheral neurons, being primarily located on presynaptic nerve terminals. These channels regulate the calcium flux required for depolarization-evoked release of a transmitter from synaptic endings. The transmission of pain signals from periphery to the central nervous system (CNS) is mediated by N-type calcium channels located in the spinal cord (Song et al., J. Med. Chem. 43:3474-3477 (2000)).
The six types of calcium channels (i.e., L, N, P, Q, R, and T) are expressed throughout the nervous system (Wallace, M. S., The Clinical Journal of Pain 16:580-585 (2000)). Voltage-sensitive calcium channels of the N-type exist in the superficial laminae of the dorsal horn and are thought to modulate nociceptive processing by a central mechanism. Blockade of the N-type calcium channel in the superficial dorsal horn modulates membrane excitability and inhibits neurotransmitter release, resulting in pain relief. Wallace (supra) suggests that based on animal models, N-type calcium channel antagonists have a greater analgesic potency than sodium channel antagonists.
N-type calcium channel blockers have usefulness for neuroprotection and analgesia. Ziconotide, which is a selective N-type calcium channel blocker, has been found to have analgesic activity in animal models and neuroprotective activity in focal and global ischemia models (Song et al., supra). Examples of known calcium channel blockers include flunarizine, fluspirilene, cilnipide, PD 157767, SB-201823, SB-206284, NNC09-0026, and PD 151307 (Hu et al, supra).
Blockade of N-type channels can prevent and/or attenuate subjective pain as well as primary and/or secondary hyperalgesia and allodynia in a variety of experimental and clinical conditions (Vanegas, H. et al, Pain 85:9-18 (2000)). N-type voltage-gated calcium channels (VGCC) play a major role in the release of synaptic mediators such as glutamate, acetylcholine, dopamine, norepinephrine, gamma-aminobutyric acid (GABA) and calcitonin gene-related peptide (CGRP).
Inhibition of voltage-gated L-type calcium channels has been shown to be beneficial for neuroprotection (Song et al., supra). Inhibition of cardiac L-type calcium channels can lead to hypotension. It is believed that a rapid and profound lowering of arterial pressure tends to counteract the neuroprotective effects of L-type calcium channel blockers. A need exists for antagonists that are selective for N-type calcium channels over L-type calcium channels to avoid potential hypotensive effects.
Movement of physiologically relevant substrates through ion channels can be traced by a variety of physical, optical, or chemical techniques (Stein, W. D., Transport and Diffusion Across Cell Membranes, 1986, Academic Press, Orlando, Fla.). Assays for modulators of ion channels include electrophysiological assays, cell-by-cell assays using microelectrodes (Wu, C.-F., Suzuki, N., and Poo, M. M. J. Neurosci 3(9):1888-99 (1983)), i.e., intracellular and patch clamp techniques (Neher, E. and Sakmann, B., Sci. Amer. 266:44-51 (1992)), and radioactive tracer ion techniques. The patch clamp and whole cell voltage clamp, current clamp, and two-electrode voltage clamp techniques require a high degree of spatial precision when placing the electrodes. Functional assays can be conducted to measure whole-cell currents with the patch clamp technique. However, the throughput is very limited in number of assays per day.
Radiotracer ions have been used for biochemical and pharmacological investigations of channel-controlled ion translocation in cell preparations (Hosford, D. A. et al, Brain Res. 516:192-200 (1990)). In this method, the cells are exposed to a radioactive tracer ion and an activating ligand for a period of time, the cells are then washed, and counted for radioactive content. Radioactive isotopes are well known (Evans, E. A., Muramtsu, M. Radiotracer Techniques and Applications, M. Dekker, New York (1977)) and their uses have permitted detection of target substances with high sensitivity. However, radioactive isotopes require many safety precautions. The use of alternative and safer non-radioactive labeling agents has thus increased in recent years.
Optical methods using fluorescence detection are suitable alternatives to the patch-clamp and radioactive tracer techniques. Optical methods permit measurement of the entire course of ion flux in a single cell as well as in groups of cells. The advantages of monitoring transport by fluorescence techniques include the high level of sensitivity of these methods, temporal resolution, modest demand for biological material, lack of radioactivity, and the ability to continuously monitor ion transport to obtain kinetic information (Eidelman, O. et al., Biophys. Acta 988:319-334 (1989)). The general principle of monitoring transport by fluorescence is based on having compartment-dependent variations in fluorescence properties associated with translocation of compounds.
Optical detection of electrical activity in nerve cells is conducted using voltage-sensitive membrane dyes and arrays of photodetectors (Grinvald, A., Annu. Rev. Neurosci. 8:263-305 (1985); Loew, L. M., and Simpson, L. L., Biopkys. J 34:353-65 (1981); Grinvald, A. et al., Biophys. J. 39:301-08 (1983); and Grinvald, A. et al., Biophys. J. 42:195-98 (1983)). Optical methods have been developed for measuring calcium ion flux (Scarpa, A., Methods of Enzymology 56:301 (1979), Academic Press, Orlando, Fla.; Tsien, R. Y., Biochemistry 19:2396 (1980); Grynkiewicz, G. et al., J. Biol. Chem. 260:3440 (1985)). The flux of calcium ions is typically monitored using calcium-sensitive fluorescent dyes, such as Fluo-3, Fluo-4, Calcium green, and others. (Molecular Probes Inc., Handbook of Fluorescent Probes and Research Chemicals, 7th ed., chapt 1, Eugene, Oreg.).
A need exists for novel assays that can identify compounds that modulate or block the movement of calcium ions through voltage-gated calcium channels, including N-type calcium channels.